1. Field of the Invention
The present invention relates generally to the fields of immunopreventive therapy and vaccine development. More particularly, it concerns the development of novel polypeptides and nucleic acids encoding such polypeptides that can be used to initiate, stimulate, and/or enhance an immune response. These polypeptides and nucleic acids encoding them can be used as adjuvants that can be used to generate more potent and robust vaccines against desired polypeptides. Additionally, these polypeptides and nucleic acids can be used to initiate or enhance an innate immune response in a subject.
The application also describes the use of dendritic cell recruitment using a novel strategy.
2. Description of Related Art
Many methodologies of medical treatment can be envisioned that will require or benefit from an ability to initiate, stimulate, and/or enhance an immune response. These methodologies include those depending upon the creation of an immune response against a desired antigenic polypeptide and those that depend upon the initiation or modulation of an innate immune response.
The use of adjuvants for immunization are well known in the art however, the challenge of developing safe and effective adjuvants is ongoing. A primary disadvantage with current adjuvants is that most are unsuitable for use in human vaccines, including especially genetic vaccines.
One of the first adjuvant developed was Freund""s complete adjuvant. This adjuvant has excellent immunopotentiating properties, however, its side effects are so severe that it renders the use of this adjuvant unacceptable in humans, and sometimes in animals. Other oil emulsions adjuvants such as Incomplete Freund""s Adjuvant (IFA); Montanide ISA (incomplete seppic adjuvant); Ribi Adjuvant System (RAS); TiterMax; and Syntex Adjuvant Formulation (SAF) are also associated with various side effects such as toxicity and inflammation. Oil based adjuvants in general are less desirable in genetic immunization; they create side effects such as visceral adhesions and melanized granuloma formations, and they cannot form a homogeneous mixture with DNA preparations such as DNA-based vaccines.
Bacterially derived adjuvants, such as MDP and lipid A are also associated with undesirable side effects. Bacterial products such as Bordetella pertussis, Corynebacterium granulosum derived P40 component, Lipopolysaccharide (LPS), Mycobacterium and its components, and Cholera toxin, are another preferred group of adjuvants however, although they may augment the immune response to other antigens they are associated with side effects such as epilepsy as in the case of B. pertussis, and varying levels of toxicity.
Mineral compounds which include aluminum phosphate or aluminum hydroxide (alum) and calcium phosphate as adjuvants may also be employed. Aluminum salt-based adjuvants (such as alum) have excellent safety records but poor efficacy with some antigens (Sjolander et al., 1998). They are the most frequently used adjuvants for vaccine antigen delivery presently. Aluminum salt-based adjuvants are generally weaker adjuvants than emulsion adjuvants. The most widely used is the antigen solution mixed form with pre-formed aluminum phosphate or aluminum hydroxide however, these vaccines are difficult to manufacture in a physico-chemically reproducible way which results in batch to batch variation of the vaccine. When used in large quantity an inflammatory reaction may occur at the site of injection which is generally resolved in a few week although chronic granulomas may occasionally form.
Other available adjuvants, are know to those skilled in the art. One such adjuvant include liposomes. Although liposomes show favorable characteristics for use in bulk vaccine preparations, the preparation proves to be rather complex for use with occasional antigens prepared for injection especially when the antigen is available in limited quantity. GerbuR adjuvant, an aqueous phase adjuvant although associated with minimal inflammatory effects may require frequent boosting to maintain high titer. Squalene also included in the group of adjuvants have been associated with the Gulf War Syndrome and include such side effects as arthritis, fibromayalgia, rashes, chronic headaches, sclerosis and non healing skin lesions to name a few.
Various polysaccharide adjuvants are also known to those skilled in the art. For example, Yin et al., (1989) describe the use of various pneumococcal polysaccharide adjuvants on the antibody responses of mice. The doses that produce optimal responses, or that otherwise do not produce suppression, as indicated in Yin et al., (1989) should be employed. Polyamine varieties of polysaccharides are particularly preferred, such as chitin and chitosan, including deacetylated chitin.
One approach to circumvent the challenges of adjuvants entails the pox viruses. Pox viruses belong to a diverse family of viruses that can affect both humans and animals. Parapoxviruses are one member of this family that is known to affect humans, and is of special interest dermatologically since they are epitheliotropic (Diven, 2001). Parapoxvirus ovis is a 150 kb double-stranded DNA virus that causes skin pock lesions in susceptible animals. Transmittance of this viral infection, though mainly affecting sheep and cattle, can occur in human handling infected animals (Johannessen et al, 1975). The infection begins at a break in the skin or inflamed hair follicle and is completely resolved in 4-6 weeks though generally no specific immune response is induced. Therefore, the caveat of this viral infection is that the animal or individual can be re-infected with no enhancement of resolution of the individual lesions in subsequent infections (McKeever et al, 1988). Furthermore, specific immunity to parapox is not acquired and proves to be a challenge since no parapox-based vaccine is available.
Live vaccines can be problematic in their own way, however. Even they can fail to shield against some diseases. Those that work can cause full-blown illness in people whose immune system is compromised, as in cancer patients undergoing chemotherapy, AIDS sufferers and the elderly. Such individuals may also contract disease from healthy people who have been inoculated recently. Moreover, weakened viruses can at times mutate in ways that restore virulence, as has happened in some monkeys given an attenuated simian form of HIV, the virus that causes AIDS. For some diseases, the risks of reversion to virulence are intolerable.
Hence, more effective adjuvants are needed that will enhance the immune response induced by gene vaccines. In order to develop such strategies there is a need to identify factors that are directly able to interact with the immune system as in the natural viral infection of parapox, thereby allowing for the identification of potential new adjuvants for genetic immunization.
The present invention overcomes the deficiencies in the art by identifying polypeptides that are useful in modulating immune responses and nucleic acid sequences that encode such polypeptides. For example, the applicants have identified specific parapox genetic sequences that can be employed in these manners.
In general embodiments the present invention provides a method of stimulating the immune system of a subject, comprising introducing an immunostimulatory polypeptide from Parapox virus into the subject. In a preferred embodiment, the polypeptide is a polypeptide of Parapox virus strain D1701 or NZ2. In further embodiments the polypeptide has a an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. In still yet a further embodiments the polypeptide is a B2WL polypeptide or a PP30 polypeptide.
In preferred embodiments, introducing the polypeptide into the subject comprises introducing a nucleic acid encoding the polypeptide into the subject. In specific embodiments, the nucleic acid encodes the polypeptide of Parapox virus strain D1701 or NZ2. In still yet a further embodiment, the nucleic acid encoding the polypeptide, has a nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO. 3, or SEQ ID NO: 5. In another embodiment the nucleic acid encoding the polypeptide, is a nucleic acid encoding a B2WL or a PP30 polypeptide.
Additionally, the present invention provides a nucleic acid comprised in an expression vector. In some embodiments, the expression vector is a linear or circular expression element, a plasmid vector, or a viral vector. The invention further contemplates the administration of the nucleic acid orally, by inhalation, or by injection into the subject. Further, the nucleic acid is injected via microprojectile bombardment.
In providing a method of stimulating the immune system of a subject, comprising introducing an immunostimulatory polypeptide from Parapox virus into the subject, the present invention further provides a method of stimulating, initiating or enhancing an innate immune response in the subject. In further embodiments, the immunostimulatory polypeptide is employed as an adjuvant to initiate stimulate, or enhance an immune response to a second polypeptide against which an immune response is desired. The invention further contemplates introducing a second polypeptide into the subject. In still yet another embodiment, the invention provides a method of introducing the second polypeptide into the subject comprising introducing a nucleic acid encoding the second polypeptide into the subject. The invention further embodies a method of genetic immunization, and a method of producing antibodies against the second polypeptide.
The nucleic acid encoding the second polypeptide against which an immune response is desired is comprised in a vector which further comprises a nucleic acid encoding the polypeptide adjuvant. The invention embodies a vector such as a linear or circular expression element, a plasmid vector, or a viral vector.
The invention embodies a method of genetic immunization further comprising enhancing an immune response in the subject such as a mammal. In another embodiment a mammal is a human or a mouse.
A method of genetic immunization comprising introducing into the subject a nucleic acid segment encoding a Parapox immunostimulatory polypeptide and a nucleic acid segment encoding a second polypeptide against which an immune response is desired is provided in the present invention. The protein expressed from the polypeptide is an immunostimulatory polypeptide having an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, and the nucleic acid segment having a sequence of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5. The protein expressed from the polypeptide is a B2WL or PP30 polypeptide further defined as an immunostimulant.
The nucleic acid segment encoding the polypeptide adjuvant is comprised in a vector such as a linear or circular expression element, a plasmid vector or a viral vector. The invention further contemplates administering a nucleic acid segment encoding the polypeptide adjuvant orally, by inhalation, or by injection. The nucleic acid segment encoding the polypeptide adjuvant is injected via microprojectile bombardment.
The nucleic acid segment encoding the second polypeptide is comprised in a vector which further comprises the nucleic acid segment encoding the immunostimulatory polypeptide. The vector of the present invention further embodies a linear or circular expression element, a plasmid vector or a viral vector. The vector comprising the second polypeptide is administered orally, by inhalation, or by injection into the subject. The vector comprising the second polypeptide is injected via microprojectile bombardment.
The invention further provides a subject such as a mammal which is further defined as a human or a mouse. In a general embodiment the invention further comprising immunizing the subject.
In a particular embodiment the invention provides a construct comprising a nucleic acid encoding a Parapox immunostimulatory polypeptide such as a polypeptide of Parapox strain D1701 or NZ2. In a particular embodiment, the construct has a sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, and the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5. In yet another particular embodiment, the Parapox immunostimulatory polypeptide is a B2WL or PP30 polypeptide.
The construct is further defined as encoding a second polypeptide against which an immune response is desired.
In a particular embodiment the construct is further defined as a vector. The construct is further defined as a linear or circular expression element, as a plasmid vector, or as a viral vector.
In a particular embodiment the invention provides a method of initiating dendritic cell accumulation with an immunostimulatory polypeptide from parapox virus, at the site of inoculation. This method is further defined as a method for treating a subject and as a method for in vitro purification.